The objective of this grant is to elucidate basic mechanisms of hemostasis and to devise strategies to alter the natural history of thrombotic disorders. There are two projects. The first deals with the role of platelets, leukocytes, and endothelial cells in the initiation and control of coagulation reactions. Prothrombin activation on platelets will be studied using homogeneous factor Va and its component peptides. The role of factor Va in protein C activation will be elucidated using the isolated factor Va peptides. The relation between thrombomodulin and factor Va, two proteins that both participate in protein C activation, will be studied using cultured endothelial cells and antibodies to thrombomodulin and factor Va. Monoclonal antibodies to protein C, factor Va and its component chains will be prepared. The platelet protease that activates factor V will be purified and its role in the initiation of platelet surface prothrombin activation will be investigated using antibodies specific for factor Va and the western blotting technique. The second project deals with the mechanism of eicosanoid precursor uptake and release by platelets and cultured cells. The fatty acid specificity of arachidonoyl CoA synthetase will be elucidated using a variety of 14C-labeled polyunsaturated fatty acids and compared to the high affinity fatty acid uptake capacity of platelets. The fatty acid specificity of the agonist-induced fatty acid release mechanism of platelets will also be elucidated. Arachidonoyl CoA synthetase will be purified to homogeneity and antibodies prepared. The technique of suicide selection after mutagenesis will be used to select arachidonate uptake and release mutants of HSD mouse fibrosarcoma cells. Mutants will be isolated by cloning and replica-plating on polyester cloth. Mutant cell lines will be useful in the elucidation of mechanisms of fatty acid uptake and release in these cells.